After years of declining case rates, tuberculosis is again a major public health threat in the United States. Serious outbreaks involving patients infected with the human immunodeficiency virus (HIV) have occurred in several major metropolitan areas. Cases have also increased in other population groups, including the homeless, prisoners, migrant farm workers, immigrants, and health care workers.
Tuberculosis (TB) is a bacterial disease caused by organisms of the M. tuberculosis complex (i.e., M. tuberculosis (MTB), M. bovis and M. africanum). It is transmitted primarily by airborne droplets produced when individuals with pulmonary or laryngeal tuberculosis sneeze, cough, or speak. In the United States, the number of tuberculosis cases reported annually declined steadily between 1953 and 1985; however, in 1986 the rate for newly diagnosed cases began to increase, with a total of 26,283 cases were reported in 1991.
The antibiotic rifampin has long been an extremely effective antimicrobial agent and is one of the two major first-line anti-tuberculosis drugs. Rifampin has a unique site of action on the beta subunit (rpoB) of prokaryotic RNA polymerase, documented both biochemically (M. E. Levin et al., Molec. Microbiol., 8, 277-285 (1993); F. G. Winder in The Biology of the Mycobacterial, Vol. 1, C. Ratledge et al., Eds., Academic Press: New York, pp. 353-438 (1982); and T. Yamada et al., Antimicrob. Agents Chemother., 27, 921-924 (1985)), and genetically (D. J. Jin et al., J. Molec. Biol., 202, 45-58 (1988); and N. Honore et al., Antimicrob. Agents Chemother., 37, 414-418 (1993)). Single site mutations in the gene for the beta subunit of RNA polymerase, rpoB, that confer rifampin resistance in Escherichia coil are well-characterized (Jin et al., J. Mol. Biol., 202, 45-48 (1988)). Mutations conferring rifampin resistance in M. tuberculosis (Telenti et al., Lancet, 341, 647-650 (1993)), Mycobacterium leprae (Honore et al., Antimicrob. Agents Chemother., 37, 414-418 (1993)), and Mycobacterium srnegmatis (Levin et al., Mol. Microbiol., 8, 277-285 (1993)) have been similarly mapped to the same region in the sequence encoding the beta subunit of RNA polymerase. The ability of single base-pair mutations in the rpoB region to confer rapidly developing high-level resistance to rifampin in E. coil is consistent with the known high frequency of developing rifampin resistance in MTB (M. Tsukamura, Tubercle, 53, 111-117 (1972)). The increasing incidence of rifampin-resistant MTB strains make it imperative to determine clinical drug susceptibility immediately upon diagnosis of TB.
Since 1990, outbreaks of multi-drug resistant tuberculosis (MDR-TB) involving over 200 patients have been reported to the Centers for Disease Control (CDC). All these outbreaks were characterized by the transmission of strains of M. tuberculosis resistant to at least isoniazid and rifampin, with some strains showing additional resistance to other drugs including ethambutol, streptomycin, ethionamide, kanamycin, and rifabutin. As used herein, MDR-MTB refers the the multi-drug resistant strains of the organism, M. tuberculosis, and MDR-TB refers to the drug-resistant disease produced by the multi-drug resistant organism. Delays in the laboratory diagnosis and reporting of drug-resistant tuberculosis contributed to the magnitude of these outbreaks since cases were not rapidly identified, the organism was not isolated, or the patients were not put on adequate therapy.
A conclusive diagnosis of tuberculosis depends on the isolation and identification of the etiologic agent, Mycobacterium tuberculosis, which generally requires 3-8 weeks. Design of an appropriate therapeutic regimen depends on the results of subsequent antituberculosis susceptibility testing by the agar dilution method and produces additional delays of 3-6 weeks (Roberts et al., "Mycobacterium" in Manual of Clinical Microbiology, 5th Ed.; A. Balows et al., Eds.; American Society for Microbiology: Washington; pp. 304-339 (1991). Identification and drug resistance testing can now also be accomplished more quickly by using the BACTEC radiometric method. (Tenover et al., J. Clin. Microbiol., 31,767-779 (1993) and Huebner et al., J. Clin. Microbiol., 31,771-775 (1993)). Acid fast bacilli are detected in the BACTEC bottle, and an identification is made using a nucleic acid hybridization technique on the BACTEC-derived growth. Drug susceptiblity testing is then conducted using the same BACTEC growth to inoculate fresh BACTEC bottles containing various antituberculous drugs. This procedure reduces the time needed to generate a complete analysis, but the total time required to report susceptibility results for MTB is still typically in excess of 20 days. The need to minimize the transmission of newly identified multi-drug resistant strains of MTB requires the development of much more rapid identification procedures.
Since rifampin resistance in MTB correlates well with multi-drug resistance (Fischl et al., Ann. Intern. Med., 117, 177-183 (1992); Frieden et al., N. Engl. J. Med., 328, 521-526 (1993); Goble et al., N. Engl. J. Med., 328, 527-532 (1993)), it can be used as a surrogate marker for MDR-MTB. Genotypic detection of multi-drug resistant MTB directly from clinical specimens is theoretically the fastest and most direct step toward determining effective therapy for patients infected with MDR-TB. A rapid test that could be performed directly on a patient specimen and that would both confirm a TB diagnosis and indicate whether it is a drug-resistant or drug-sensitive strain would be a major advance.